Carbohydrate fatty acid monosulphate ester is a potent adjuvant for low-dose seasonal influenza vaccines.

There is still a need for a better and affordable seasonal influenza vaccine and the use of an adjuvant could solve both issues. Therefore, immunogenicity of a combination of low dose of 1/5TH (3 µg of HA) a licensed seasonal flu vaccine with the novel carbohydrate fatty acid monosulfate ester (CMS)-based adjuvant was investigated in ferrets and safety in rabbits. Without CMS, hemagglutination inhibition (HI) antibody titers ranged from ≤5 to 26 three weeks post immunization 1 (PV-1) and from 7 to 134 post-immunization 2 (PV-2) in ferrets. Virus neutralizing (VN) antibody titers ranged from 20 to 37 PV-1 and from 21 to 148 PV-2. CMS caused 10 to 111- fold increase in HI titers and 3 to 58- fold increase in VN titers PV-1 and PV-2, depending on influenza strain and dose of adjuvant. Eight mg of CMS generated significantly higher antibody titers than 1 or 4 mg, while 1 and 4 mg induced similar responses. Three µg of HA plus 4 mg of CMS was considered the highest human dose and safety of two-fold this dose was determined in acute and repeated-dose toxicity studies in rabbits conducted according to OECD GLP guidelines. The test item did not elicit any clinical signs, local reactions, effect on body weight, effect on urine parameters, effect on blood biochemistry, or gross pathological changes. In blood, increased numbers of neutrophils, lymphocytes and/or monocytes were noted and in iliac lymph nodes, increased cellularity of macrophages of minimal to mild degree were observed. In both ferrets and rabbits, body temperature increased with increasing dose of CMS to a maximum of 1 ˚C during the first day post-immunization, which returned to normal values during the second day. In the local tolerance study, histopathology of the site of injection at 7 days PV-1 revealed minimal, mild or moderate inflammation in 5, 8 and 5 animals, respectively. In the repeated-dose study and 21 days PV-3, minimal, mild or moderate inflammation was observed in 15, 18 and 3 animals, respectively. We concluded that the data show CMS is a potent and safe adjuvant ready for further clinical development of a seasonal influenza vaccine and combines high immunogenicity with possible antigen-sparing capacity.

Challenging development of storable particles for oral delivery of a physiological nitric oxide donor.

Nitric oxide (NO) deficiency is often associated with several acute and chronic diseases. NO donors and especially S-nitrosothiols such as S-nitrosoglutathione (GSNO) have been identified as promising therapeutic agents. Although their permeability through the intestinal barrier have recently be proved, suitable drug delivery systems have to be designed for their oral administration. This is especially challenging due to the physico-chemical features of these drugs: high hydrophilicity and high lability. In this paper, three types of particles were prepared with an Eudragit® polymer: nanoparticles and microparticles obtained with a water-in-oil-in-water emulsion/evaporation process versus microparticles obtained with a solid-in-oil-in-water emulsion/evaporation process. They had a similar encapsulation efficiency (around 30%), and could be freeze-dried then be stored at least one month without modification of their critical attributes (size and GSNO content). However, microparticles had a slightly slower in vitro release of GSNO than nanoparticles, and were able to boost by a factor of two the drug intestinal permeability (Caco-2 model). Altogether, this study brings new data about GSNO intestinal permeability and three ready-to-use formulations suitable for further preclinical studies with oral administration.

Wheat digalactosyldiacylglycerol molecular species profiling using porous graphitic carbon stationary phase.

The potential of porous graphitic carbon stationary phase (PGC) was assessed for the separation of molecular species of digalactosyldiacylglycerol (DGDG). Detection was by an evaporative light scattering detector (ELSD). A conventional optimization strategy allowed definition of a quaternary non-aqueous mobile phase and separation of 9 wheat DGDG molecular species with isocratic elution: methanol/toluene/tetrahydrofuran/chloroform 64.3/21.5/13.7/0.5 v/v with 0.1% of triethylamine and a stoichiometric amount of formic acid. The molecular species were identified by LC/MS. The chromatographic behavior of DGDG on PGC was then compared to previous studies. The addition of a carbon double bond on the alkyl chain decreased the retention. This contribution was less important when the number of unsaturations increased in the alkyl chain. The consequence of this retention behavior with PGC was an elution order of molecular species which did not agree with the partition number as observed with C18 grafted stationary phases.

Glycolipid class profiling by packed-column subcritical fluid chromatography.

The potential of packed-column subcritical fluid chromatography (SubFC) for the separation of lipid classes has been assessed in this study. Three polar stationary phases were checked: silica, diol, and poly(vinyl alcohol). Carbon dioxide (CO2) with methanol as modifier was used as mobile phase and detection performed by evaporative light scattering detection. The influence of methanol content, temperature, and pressure on the chromatographic behavior of sphingolipids and glycolipids were investigated. A complete separation of lipid classes from a crude wheat lipid extract was achieved using a modifier gradient from 10 to 40% methanol in carbon dioxide. Solute selectivity was improved using coupled silica and diol columns in series. Because the variation of eluotropic strength depending on the fluid density changes, a normalized separation factor product (NSP) was used to select the nature, the number and the order of the columns to reach the optimum glycolipid separation.

Mechanism of response enhancement in evaporative light scattering detection with the addition of triethylamine and formic acid.

Triethylamine with an equimolar amount of formic acid added to the mobile phase provides an enhancement of the evaporative light scattering detector (ELSD) response. After characterization of the influence of various parameters on the ELSD response, a sequential strategy was defined to elucidate this response enhancement. The response enhancement was more marked at low mobile phase flow rate, and was highly dependent on solutes and solvents. The influence of drift tube temperature on response enhancement with various solutes demonstrated that triethylamine and formic acid mainly act as mass amplifiers by the inclusion of triethylamine-formic acid clusters inside the droplets.